Colorectal carcinoma is the second most common cause of cancer death in the United States with 102,900 new cases in 2010. Over 90% of colorectal cancer deaths occur in people over 50 years of age which constitutes a large population of Veterans. Standard therapy for metastatic colon cancer results in a median survival of 22 months. Since conventional therapy is relatively non-specific and cytotoxicity occurs in both tumor and normal cells, there is a great need for selective targeting of colorectal cancer with sparing of normal cells. This project will test our hypothesis that design of targeted therapy for colorectal cancer is possible by a two-step approach which combines gene therapy to enhance metabolism of a pro-drug, Tiazofurin within cancer cells following therapy. Our previous studies have shown that Tiazofurin is converted to an active form, Thiazole-4-carboxamide Adenine Dinucleotide (TAD) by the enzyme Nicotinamide 5'-Mononucleotide Adenylyltransferase (NMNAT). TAD is a potent inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH) which leads to a cessation of guanylate synthesis (including GTP and dGTP) and cancer cell death. In the Progress Report we have determined in human colorectal cancer cell lines that there was a good correlation between NMNAT expression and cell-kill by Tiazofurin. We have cloned the human NMNAT2 gene that is expressed in the cytoplasm, transfected it into colorectal cancer cells and demonstrated that cell-kill is enhanced with Tiazofurin. We have further increased the specificity of targeting exploiting folate receptors (FR) which are over-expressed on the surface of colorectal cancer cells. This was achieved by designing and synthesizing folate-tethered nanoparticles encapsulating Tiazofurin, so that these are taken up only by cancer cells that over-express FR. Using this strategy, we demonstrated enhancement of cell-kill by Tiazofurin. We have also constructed adeno-associated virus2 (AAV2) bearing hNMNAT2, encapsulated these AAV2 in folate- tethered nanoparticles and demonstrated successful expression of hNMNAT2 gene into colorectal cancer cells. To further increase the specificity, we will take advantage of the expression of carcinoembryonic antigen (CEA) by colorectal cancer cells and have designed an AAV2 vector where hNMNAT2 is driven by CEA. We are now poised to conduct proof-of-principle studies in vitro that can then be applied to studies in mice. The specific objectives of the proposal are: Specific Aim 1: To demonstrate preferential FR-mediated uptake of folate-tethered nanoparticles that contain AAV2 viruses bearing CEA-promoter-driven-hNMNAT2 into cultured colorectal cancer cells when compared to normal colonic cells; and determine that over-expression of hNMNAT2 (by CEA containing colorectal cancer cells) renders them more susceptible to Tiazofurin-mediated suicide. Specific Aim 2: To assess the extent of selectivity of targeting human colorectal tumors (that express FR-, CEA-, and hNMNAT2), we will first individually clone specialized Luciferase (Luc) reporter genes in AAV2 (AAV2-Luc, AAV2-CEA-Luc and AAV2-hNMNAT2-Luc); then we will inject these viruses into mice and assess the expression of Luc using dynamic bioluminescent image analysis in various mouse tissues. Specific Aim 3: To assess toxicity of targeting FR, CEA, and hNMNAT-2 in mouse tissues using dynamic bioluminescent image analysis after transducing AAV2-CEA-hNMNAT-2-Luc viruses into mice and treating with Tiazofurin in folate-tethered nanoparticles. Specific Aim 4: To determine if the use of a gene delivery system (FR-CEA- hNMNAT2) that leads to the selective over-expression of hNMNAT2 in colorectal tumors in mice will lead to enhanced tumor cell-kill with folate-tethered nanoparticles containing Tiazofurin. POTENTIAL IMPACT ON VETERANS HEALTH CARE: The potential significance of these new proofs-of- principle studies is that they will set the stage for the next level of studies that could culminate in a new form of gene-directe enzyme pro-drug therapy for colorectal cancer. Therefore, this grant proposal is highly relevant to Veterans Health Care since colorectal cancer is estimated to claim 50,000 Veteran lives in 2010.